Zoonotic viral infections are responsible for a large portion of emerging infectious diseases in humans. However, for many viruses the molecular basis that determines host range, virulence and the potential for cross-species transmission is poorly understood. The long-term goal is to delineate key factors that contribute to viral host range and virulence by analyzing the interaction of viruses with the host innate immune system. A major focus are model poxviruses, which display extreme differences in host range and virulence and include major human and animal pathogens. The objective of this application is to determine the contribution of species-specific interactions of the host antiviral protein kinase R (PKR) with virus inhibitors for virus replication, virulence and host range. The central hypothesis is that both viral host range and virus replication are directly influenced by the interaction of PR and its viral inhibitors. The rationale of this project is that an improved understanding of the mechanisms that determine virus host range can ultimately result in a better prediction of host switches and changes in virulence and also might lead to new alternative strategies for disease control. Based on strong preliminary data, the main hypothesis will be tested by pursuing three specific aims: 1) Characterize host-specific PKR inhibition by poxvirus inhibitors; 2) Elucidate the molecular basis for PKR sensitivity to viral inhibitors; and 3) Evaluate the effects and functional consequences of naturally occurring variants of rabbit PKR and myxoma virus inhibitors. Under the first aim, the interactions between PKR proteins from different host species with PKR inhibitors from prototypic poxviruses will be studied and their functional relevance in cell line infection assays tested. In the second aim, the molecular basis for species-specific PKR-inhibitor interactions will be determined by combining mutagenesis, binding and infection assays and the potential of PKR to resist PKR inhibitors from other viruses will be tested. Under the third aim, naturally evolved variants of PKR from rabbits and from myxoma virus PKR inhibitors, as well as mutants that were generated in artificial evolution experiments, will be analyzed for effects in interaction and infection assays to test the PKR-inhibitor interactions in textbook example for host-virus coevolution. The proposed research is significant because it will lead to the better understanding of the molecular basis for poxvirus host range, yield novel insights into the mechanisms of resistance to viruses and how they evolve, and might lead to a better prediction of which viruses have a strong potential for cross-species transmission. This project is innovative because it introduces new strategies and novel tools to elucidate the importance of host-virus interactions for virus replication and host range and explores the novel concept that the outcome of cross- species virus transmission is strongly influenced by chance, but might be predicted by experimental analyses. Ultimately, the knowledge gained during this project will lead to the better understanding of the molecular mechanisms behind virus host range and resistance and is expected to propel the field forward.